The inability to transduce a sufficient fraction of the stem cell pool is a major limitation to gene therapy for sickle cell disease. In this project we attempt to overcome this obstacle by: a) identifying the optimal conditions for the transduction of human hemopoietic stem cells in vitro. Novel cytokines, such as flk-2 ligand, will be tested in our transduction protocols, as will the effects of substrata (CS-1, VCAM-1, or fibronectin), which may provide concentrated foci of retroviral particles. Due to the similarity between human and baboon hemopoiesis, transduction conditions that are optimal in humans will be applied to our primate model; b) Optimal mobilization conditions will be identified in primates so that the greatest possible number of stem cells can be harvested for ex vivo manipulation; c) Repeated cycles of mobilization and reinfusion will be performed using a vector containing a dominant selectable marker. In vivo selection will be applied using a drug to which cells containing the marker are resistant. A schedule of drug administration will be used that is intended to simultaneously provide mild conditioning and in vivo selection; d) As a control for conditioning and selection in c), repeated cycles of mobilization, transduction, and reinfusion, without myeloablation or selection will be performed; e) The relative efficiencies of MLV- versus AAV- based vectors to transduce long-term repopulating cells will be compared in individual animals; and f) the efficacy and safety of a new class of vectors based on human foamy virus and lentivirus will be tested.